CN109266552B - A microfluidic chip simulating tissue microenvironment and a method for inducing stem cell specific differentiation using the same - Google Patents
A microfluidic chip simulating tissue microenvironment and a method for inducing stem cell specific differentiation using the same Download PDFInfo
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- CN109266552B CN109266552B CN201811429374.XA CN201811429374A CN109266552B CN 109266552 B CN109266552 B CN 109266552B CN 201811429374 A CN201811429374 A CN 201811429374A CN 109266552 B CN109266552 B CN 109266552B
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- 210000000130 stem cell Anatomy 0.000 title claims abstract description 26
- 238000000034 method Methods 0.000 title claims abstract description 15
- 230000001939 inductive effect Effects 0.000 title claims abstract description 9
- 230000004069 differentiation Effects 0.000 title claims description 13
- 230000010412 perfusion Effects 0.000 claims abstract description 188
- 239000002121 nanofiber Substances 0.000 claims abstract description 62
- 239000007788 liquid Substances 0.000 claims abstract description 15
- 230000000638 stimulation Effects 0.000 abstract description 16
- 239000003814 drug Substances 0.000 abstract description 2
- 210000002435 tendon Anatomy 0.000 description 5
- 210000004027 cell Anatomy 0.000 description 4
- 239000012528 membrane Substances 0.000 description 4
- 210000001074 muscle attachment cell Anatomy 0.000 description 4
- 238000000338 in vitro Methods 0.000 description 3
- 210000004271 bone marrow stromal cell Anatomy 0.000 description 2
- 239000011159 matrix material Substances 0.000 description 2
- 210000002901 mesenchymal stem cell Anatomy 0.000 description 2
- 230000008569 process Effects 0.000 description 2
- 230000001105 regulatory effect Effects 0.000 description 2
- 238000011160 research Methods 0.000 description 2
- 102000008186 Collagen Human genes 0.000 description 1
- 108010035532 Collagen Proteins 0.000 description 1
- 230000032683 aging Effects 0.000 description 1
- 230000006907 apoptotic process Effects 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- 230000003592 biomimetic effect Effects 0.000 description 1
- 230000024245 cell differentiation Effects 0.000 description 1
- 230000001413 cellular effect Effects 0.000 description 1
- 229920001436 collagen Polymers 0.000 description 1
- 230000001276 controlling effect Effects 0.000 description 1
- 238000007796 conventional method Methods 0.000 description 1
- 239000012531 culture fluid Substances 0.000 description 1
- 238000010586 diagram Methods 0.000 description 1
- 238000005516 engineering process Methods 0.000 description 1
- 239000000835 fiber Substances 0.000 description 1
- 239000012530 fluid Substances 0.000 description 1
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- 210000004523 ligament cell Anatomy 0.000 description 1
- 230000004060 metabolic process Effects 0.000 description 1
- 230000001766 physiological effect Effects 0.000 description 1
- 230000035790 physiological processes and functions Effects 0.000 description 1
- 238000005086 pumping Methods 0.000 description 1
- 238000012216 screening Methods 0.000 description 1
- 230000019491 signal transduction Effects 0.000 description 1
- 238000004088 simulation Methods 0.000 description 1
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- C12M29/00—Means for introduction, extraction or recirculation of materials, e.g. pumps
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Abstract
The invention relates to a micro-fluidic chip simulating a tissue microenvironment and a method for inducing stem cells to differentiate specifically by using the micro-fluidic chip, belonging to the field of medicine. The microfluidic chip comprises a perfusion culture channel layer, a nanofiber-coated film layer and a gas channel layer, wherein the perfusion culture channel layer is arranged on one side of the perfusion culture channel layer, the gas channel layer is provided with the gas channel on one side of the nanofiber-coated film layer, the nanofiber-coated film layer is arranged between the perfusion culture channel layer and the gas channel layer, the nanofiber in the nanofiber-coated film layer is in contact with one side of the perfusion culture channel, the film in the nanofiber-coated film layer is in contact with one side of the gas channel layer, the gas channel passes through the perfusion culture channel and is perpendicular to the perfusion culture channel, and the gas flow direction in the gas channel is perpendicular to the liquid flow direction in the perfusion culture channel. The air passage passes through the perfusion culture passage, and the air pressure deforms the film layer covered with the nano fibers, so that tension stimulation is realized.
Description
Technical Field
The invention relates to a micro-fluidic chip simulating a tissue microenvironment and a method for inducing stem cells to differentiate specifically by using the micro-fluidic chip, belonging to the field of medicine.
Background
Tissue microenvironment simulation is an important means for inducing stem cell specific differentiation, and a dynamically coordinated three-dimensional microenvironment plays an important role in regulating and controlling the growth and differentiation of stem cells. In recent years, more and more researches show that the mechanical microenvironment and the mechanical signal transduction play an important role in the physiological processes of cells such as stem cell self-renewal, differentiation, aging, apoptosis and the like, and because of the specificity of tendon structures, a small number of tenocytes are always in a mechanical load state and are arranged among collagen fibers along the longitudinal axis, and the metabolism and various physiological activities of tendons are closely related to the stimulation of the mechanical physical microenvironment. Many studies currently suggest that mesenchymal stem cells (MESENCHYMAL STEM CELLS, MSCs) are capable of induced differentiation into tenocytes in vitro, and that biomimetic matrix structure stimulation or mechanical traction tension stimulation helps stem cell differentiation into tenocytes. However, the mechanical microenvironment of tendon/ligament cells in the human body is a complex integrated system consisting of a series of multifactorial components, involving aspects of matrix structural stimulation, mechanical force stimulation, interstitial flow, etc.
The mechanical microenvironment stimulus plays an important role in the process of inducing the differentiation of MSCs into tendons. However, the stimulation intensity of shear flow, the stimulation mode of fluid, the stimulation intensity of mechanical traction force, the stimulation frequency and other optimal mechanical microenvironment parameters in the process of differentiation to tenocytes are not completely known. Therefore, if the optimal microenvironment condition for stem cell directional differentiation can be selected, the method has important significance for improving the differentiation efficiency of stem cells to tendons.
The cellular microenvironment is a complex set of multifactorial and space-time variable that plays a decisive role in the behavior and function of the cells. However, it is difficult to provide such a complex, micro-scale growth environment for cells in vitro with conventional methods of cell biology, so that many in vitro research results are far from in vivo.
Disclosure of Invention
According to the invention, the perfusion culture channel layer, the film layer coated with the nanofibers and the air channel layer are integrated, so that not only is the stimulation of culture solutions at different angles to stem cells realized, but also the stimulation of different deformations of the film to the stem cells is realized, and the problems are solved.
The invention provides a micro-fluidic chip for simulating a tissue microenvironment, which comprises a perfusion culture channel layer, a film layer covered with nanofibers and an air channel layer; one surface of the perfusion culture channel layer is provided with a perfusion culture channel; one surface of the air passage layer is provided with an air passage; the film layer covered with the nano fibers is arranged between the perfusion culture channel layer and the air channel layer, so that the nano fibers in the film layer covered with the nano fibers are contacted with one surface of the perfusion culture channel layer, on which the perfusion culture channel is arranged, and the film in the film layer covered with the nano fibers is contacted with one surface of the air channel layer, on which the air channel is arranged; the gas path channel passes through the perfusion culture channel and is perpendicular to the perfusion culture channel, so that the gas flow direction in the gas path channel is perpendicular to the liquid flow direction in the perfusion culture channel.
In the invention, one surface of the perfusion culture channel layer is preferably provided with a plurality of perfusion culture channels; the gas channel passes through each perfusion culture channel and is perpendicular to each perfusion culture channel, so that the gas flow direction in the gas channel is perpendicular to the liquid flow direction in each perfusion culture channel, and the width of the gas channel passing through each perfusion culture channel is different.
The invention preferably uses the film layer covered with the nano-fibers as a plurality of nano-fibers which are arranged on the film in parallel; different perfusion culture channels and the nanofibers form different angles, so that the liquid flowing directions in the different perfusion culture channels and the axial directions of the nanofibers form different angles.
The invention is preferable that one surface of the perfusion culture channel layer is provided with a plurality of groups of perfusion culture channels, and each group comprises a plurality of perfusion culture channels; the same group of perfusion culture channels and the nanofibers form the same angle, so that the liquid flowing direction in the same group of perfusion culture channels and the axial direction of the nanofibers form the same angle; the perfusion culture channels of different groups form different angles with the nanofiber, so that the liquid flowing directions in the perfusion culture channels of different groups form different angles with the axial direction of the nanofiber; the gas path channels pass through the perfusion culture channels and are perpendicular to the perfusion culture channels, so that the gas flow direction in the gas path channels is perpendicular to the liquid flow direction in the perfusion culture channels.
The invention preferably has the advantages that the widths of the air channel channels passing through the perfusion culture channels in the same group are different.
The invention also provides a method for inducing stem cells to differentiate specifically by using the microfluidic chip, which is characterized by comprising the following steps: the method comprises the following steps: firstly, adding stem cells into each perfusion culture channel, enabling the stem cells to be on the nanofibers in the nanofiber-covered film layer, then pumping culture solution into each perfusion culture channel for perfusion culture, and then introducing air into the air channel, so that the nanofiber-covered film layer deforms to different degrees.
The invention has the beneficial effects that:
① According to the invention, the perfusion culture channels and the nanofibers are designed, so that the flowing direction of the culture fluid and the axial direction of the nanofibers form different angles, microfluidics with different angles generate different stimulation on stem cells on the nanofibers, and meanwhile, the air channel passes through the same group of perfusion culture channels, the width of each perfusion culture channel is different, and different air pressures enable the film layers covered with the nanofibers to deform to different degrees, so that tension stimulation with different intensities is realized.
② The microfluidic chip fully utilizes the integrated characteristic of a microfluidic technology platform to form an effective method for efficiently regulating the mechanical microenvironment of stem cells, and a menu-type culture scheme can be found out by using a small amount of stem cells, so that a novel technical method and an intervention target point are provided for optimizing and screening the microenvironment of stem cells differentiated to tendinous lines and improving the differentiation efficiency of the stem cells.
Drawings
In the present invention of figure 6,
FIG. 1 is a schematic plan view of a micro-fluidic chip for simulating a tissue microenvironment according to example 1;
fig. 2 is a schematic structural diagram of a micro-fluidic chip simulating a tissue microenvironment according to embodiment 1;
FIG. 3 is a schematic view of the structure of the nanofiber coated membrane layer in perfusion culture channel I after air is introduced in example 2;
FIG. 4 is a schematic view of the structure of the nanofiber coated membrane layer in perfusion culture channel II after air is introduced in example 2;
FIG. 5 is a schematic view of the structure of the nanofiber coated membrane layer in perfusion culture channel III after air is introduced in example 2;
FIG. 6 is a schematic view of the structure of the nanofiber coated membrane layer in perfusion culture channel IV after air is introduced in example 2;
the device comprises a perfusion culture channel layer (1), a perfusion culture channel layer (101), a perfusion culture channel (I, 102), a perfusion culture channel (II, 103), a perfusion culture channel (III, 104), a perfusion culture channel (IV, 105), a perfusion culture channel (V, 106), a perfusion culture channel (VI, 107), a perfusion culture channel (VII, 108), a perfusion culture channel (VIII, 109), a perfusion culture channel (IX, 110), a perfusion culture channel (X, 111), a perfusion culture channel (XI, 112), a perfusion culture channel (XII, 2), a nanofiber-coated film layer, 3, an air channel layer (301) and an air channel (II, 103).
Detailed Description
The following non-limiting examples will enable those of ordinary skill in the art to more fully understand the invention and are not intended to limit the invention in any way.
Example 1
As shown in fig. 1 and 2, the microfluidic chip comprises a perfusion culture channel layer 1, a film layer 2 covered with nanofibers and a gas path channel layer 3;
One surface of the perfusion culture channel layer 1 is provided with 3 groups of perfusion culture channels with the same shape;
Group 1 includes perfusion culture channel I101, perfusion culture channel II 102, perfusion culture channel III 103, and perfusion culture channel IV 104;
group 2 includes perfusion culture channel V105, perfusion culture channel VI 106, perfusion culture channel VII 107, and perfusion culture channel VIII 108;
group 3 includes perfusion culture channel IX 109, perfusion culture channel X110, perfusion culture channel XI 111, perfusion culture channel XII 112;
The film layer 2 covered with the nano-fibers is formed by arranging a plurality of nano-fibers on the film in parallel;
one surface of the air channel layer 3 is provided with an air channel 301;
the film layer 2 covered with the nano fibers is arranged between the perfusion culture channel layer 1 and the air channel layer 3, so that the nano fibers in the film layer 2 covered with the nano fibers are contacted with one surface of the perfusion culture channel layer 1, on which the perfusion culture channel is arranged, and the film in the film layer 2 covered with the nano fibers is contacted with one surface of the air channel layer 3, on which the air channel 301 is arranged;
The perfusion culture channel I101, the perfusion culture channel II 102, the perfusion culture channel III 103 and the perfusion culture channel IV 104 form an angle of 0 degree with the nanofiber, so that the liquid flow directions in the perfusion culture channel I101, the perfusion culture channel II 102, the perfusion culture channel III 103 and the perfusion culture channel IV 104 form an angle of 0 degree with the axial direction of the nanofiber;
The perfusion culture channel V105, the perfusion culture channel VI 106, the perfusion culture channel VII 107 and the perfusion culture channel VIII 108 form an angle of 45 degrees with the nanofiber, so that the liquid flowing directions in the perfusion culture channel V105, the perfusion culture channel VI 106, the perfusion culture channel VII 107 and the perfusion culture channel VIII 108 form an angle of 45 degrees with the axial direction of the nanofiber;
The perfusion culture channel IX 109, the perfusion culture channel X110, the perfusion culture channel XI 111 and the perfusion culture channel XII 112 are all at an angle of 90 degrees with the nanofiber, so that the liquid flowing directions in the perfusion culture channel IX 109, the perfusion culture channel X110, the perfusion culture channel XI 111 and the perfusion culture channel 112 are at an angle of 90 degrees with the axial direction of the nanofiber;
the gas path channel 301 passes through and is perpendicular to each group of perfusion culture channels, so that the gas flow direction in the gas path channel 301 is perpendicular to the liquid flow direction in each group of perfusion culture channels;
The width of the air channel 301 passing through the perfusion culture channel I101 is larger than the width of the air channel 301 passing through the perfusion culture channel II 102 is larger than the width of the air channel 301 passing through the perfusion culture channel III 103 is larger than the width of the air channel 301 passing through the perfusion culture channel IV 104;
The width of the air path channel 301 passing through the perfusion culture channel v 105 = the width of the air path channel 301 passing through the perfusion culture channel ix 109 = the width of the air path channel 301 passing through the perfusion culture channel i 101;
The width of the air path channel 301 passing through the perfusion culture channel vi 106 = the width of the air path channel 301 passing through the perfusion culture channel x 110 = the width of the air path channel 301 passing through the perfusion culture channel ii 102;
the width of the air path channel 301 through the perfusion culture channel vii 107 = the width of the air path channel 301 through the perfusion culture channel xi 111 = the width of the air path channel 301 through the perfusion culture channel iii 103;
The width of the air path channel 301 passing through the perfusion culture channel viii 108 = the width of the air path channel 301 passing through the perfusion culture channel xii 112 = the width of the air path channel 301 passing through the perfusion culture channel iv 104.
Example 2
A method of inducing stem cell specific differentiation using the microfluidic chip of example 1, the method comprising:
The stem cells are added into each perfusion culture channel, so that the stem cells are on the nanofibers in the nanofiber-covered film layer 2, then the culture solution is pumped into each perfusion culture channel for perfusion culture, the stimulation of the culture solution with different angles to the nanofibers arranged in parallel is formed, and then air is introduced into the air channel 301, as shown in fig. 3, 4, 5 and 6, so that the nanofiber-covered film layer 2 deforms to different degrees, and tension stimulation with different intensities is realized.
Claims (2)
1. The micro-fluidic chip simulating the tissue micro-environment is characterized by comprising a perfusion culture channel layer (1), a film layer (2) covered with nano fibers and a gas channel layer (3);
One surface of the perfusion culture channel layer 1 is provided with 3 groups of perfusion culture channels with the same shape;
Group 1 includes perfusion culture channel I (101), perfusion culture channel II (102), perfusion culture channel III (103), and perfusion culture channel IV (104);
group 2 includes perfusion culture channel V (105), perfusion culture channel VI (106), perfusion culture channel VII (107), perfusion culture channel VIII (108);
Group 3 includes perfusion culture channel IX (109), perfusion culture channel X (110), perfusion culture channel XI (111), and perfusion culture channel XII (112);
the film layer (2) covered with the nano fibers is formed by arranging a plurality of nano fibers on the film in parallel;
one surface of the air channel layer (3) is provided with an air channel (301);
The film layer (2) covered with the nano fibers is arranged between the perfusion culture channel layer (1) and the air channel layer (3), so that the nano fibers in the film layer (2) covered with the nano fibers are contacted with one surface of the perfusion culture channel layer (1) where the perfusion culture channel is arranged, and the film in the film layer (2) covered with the nano fibers is contacted with one surface of the air channel layer (3) where the air channel (301) is arranged;
the perfusion culture channel I (101), the perfusion culture channel II (102), the perfusion culture channel III (103) and the perfusion culture channel IV (104) form an angle of 0 degrees with the nanofiber, so that the liquid flowing directions in the perfusion culture channel I (101), the perfusion culture channel II (102), the perfusion culture channel III (103) and the perfusion culture channel IV (104) form an angle of 0 degrees with the axial direction of the nanofiber;
The perfusion culture channel V (105), the perfusion culture channel VI (106), the perfusion culture channel VII (107) and the perfusion culture channel VIII (108) form an angle of 45 degrees with the nanofiber, so that the liquid flowing directions in the perfusion culture channel V (105), the perfusion culture channel VI (106), the perfusion culture channel VII (107) and the perfusion culture channel VIII (108) form an angle of 45 degrees with the axial direction of the nanofiber;
The perfusion culture channel IX (109), the perfusion culture channel X (110), the perfusion culture channel XI (111) and the perfusion culture channel XII (112) form an angle of 90 degrees with the nanofiber, so that the liquid flowing directions in the perfusion culture channel IX (109), the perfusion culture channel X (110), the perfusion culture channel XI (111) and the perfusion culture channel XII (112) form an angle of 90 degrees with the axial direction of the nanofiber;
The gas path channels (301) pass through and are perpendicular to the groups of perfusion culture channels, so that the gas flow direction in the gas path channels (301) is perpendicular to the liquid flow direction in the groups of perfusion culture channels;
The width of the air channel (301) passing through the perfusion culture channel I (101) is larger than the width of the air channel (301) passing through the perfusion culture channel II (102), the width of the air channel (301) passing through the perfusion culture channel III (103) is larger than the width of the air channel (301) passing through the perfusion culture channel IV (104);
the width of the air channel (301) passing through the perfusion culture channel v (105) =the width of the air channel (301) passing through the perfusion culture channel ix (109) =the width of the air channel (301) passing through the perfusion culture channel i (101);
The width of the air channel (301) passing through the perfusion culture channel vi (106) =the width of the air channel (301) passing through the perfusion culture channel x (110) =the width of the air channel (301) passing through the perfusion culture channel ii (102);
The width of the air channel (301) passing through the perfusion culture channel vii (107) =the width of the air channel (301) passing through the perfusion culture channel xi (111) =the width of the air channel (301) passing through the perfusion culture channel iii (103);
the width of the air channel (301) passing through the perfusion culture channel viii (108) =the width of the air channel (301) passing through the perfusion culture channel nip (112) =the width of the air channel (301) passing through the perfusion culture channel iv (104).
2. A method for inducing stem cell specific differentiation by using the microfluidic chip according to claim 1, which is characterized in that: the method comprises the following steps:
Firstly, stem cells are added into each perfusion culture channel, so that the stem cells are on the nanofibers in the nanofiber-coated film layer (2), then, culture solution is pumped into each perfusion culture channel for perfusion culture, and then, air is introduced into the air channel (301), so that the nanofiber-coated film layer (2) deforms to different degrees.
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| CN101663585A (en) * | 2008-02-29 | 2010-03-03 | 亚州大学校产学协力团 | A cell chip and an automatic control system that can detect optimal conditions for stem cell differentiation using mechanical stimulation |
| CN209193992U (en) * | 2018-11-27 | 2019-08-02 | 大连医科大学附属第一医院 | A kind of micro-fluidic chip of simulated tissue microenvironment |
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| CN105624037A (en) * | 2014-11-06 | 2016-06-01 | 中国科学院大连化学物理研究所 | Method for establishing in-vitro blood-brain barrier model based on micro-fluidic chip |
| CN105733943A (en) * | 2014-12-11 | 2016-07-06 | 中国科学院大连化学物理研究所 | Three-dimensional cell microsphere cultivation and controllable release method based on micro-fluidic chip |
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| CN209193992U (en) * | 2018-11-27 | 2019-08-02 | 大连医科大学附属第一医院 | A kind of micro-fluidic chip of simulated tissue microenvironment |
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